The objectives of this proposal are to determine how protein kinase Cepsilon (PKCepsilon) signals tumor necrosis factor alpha (TNFalpha) release and whether this TNFalpha release mediates the development of metastatic squamous cell carcinoma (mSCC) in PKCepsilon-overexpressing transgenic mice. PKC is not only the major intracellular receptor for the mouse skin tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) but also is activated by a variety of stress factors including ultraviolet irradiation (UVR). PKCepsilon is among the six PKC isoforms (alpha, delta, epsilon, eta, mu, delta) expressed in the mouse skin. To determine the in vivo functional specificity of PKCe in mouse skin carcinogenesis, we generated PKCepsilon transgenic mouse (FVB/N) lines 224 and 215 that overexpress approximately 8- and 18-fold respectively PKCepsilon protein over endogenous levels in basal epidermal cells. PKCepsilon transgenic mice were observed to be highly sensitive to the development of mSCC elicited either by the repeated exposure to UVR or by the DMBA (100 nmol) - TPA (5nmol) tumor promotion protocol. During studies to find clues about how PKCe overexpression mediates development of mSCC, we found that PKCepsilon transgenic mice have dramatically elevated TNFalpha serum levels relative to their wild-type littermates either following UVR exposure or during skin tumor promotion by TPA. TPA-stimulated serum TNFalpha levels were proportional to the level of expression of epidermal PKCe in transgenic mouse lines 224 and 215. Preliminary results indicate that mouse keratinocytes are the primary source of serum TNFalpha levels, and TPA affects ectodomain shedding of TNFalpha. The release of soluble TNFalpha is catalyzed by the TNFalpha converting enzyme (TACE), a transmembrane metalloproteinase. We hypothesize that: 1) UVR- or TPA-stimulated TNFalpha release is the key downstream component of the PKCepsilon signal transduction pathway to development of mSCC in PKCe transgenic mice and 2) PKCepsilon mediates UVR/TPA-stimulated TNFalpha release by regulating TACE activity. We propose two specific aims to test this hypothesis: Specific Aim #1: To determine whether TNFalpha is essential for the development of mSCC in PKCepsilon transgenic mice. Specific Aim #2: To determine how PKCe mediates UVR- or TPA-stimulated release of TNFalpha.